RESUMEN
Stem cells generate many differentiated, short-lived cell types, such as blood, skin, and sperm, throughout adult life. Stem cells maintain a long-term capacity to divide, producing daughter cells that either self-renew or initiate differentiation. Although the surrounding microenvironment or "niche" influences stem cell fate decisions, few signals that emanate from the niche to specify stem cell self-renewal have been identified. Here we demonstrate that the apical hub cells in the Drosophila testis act as a cellular niche that supports stem cell self-renewal. Hub cells express the ligand Unpaired (Upd), which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in adjacent germ cells to specify self-renewal and continual maintenance of the germ line stem cell population.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Células Germinativas/fisiología , Glicoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Células Madre/fisiología , Transactivadores/metabolismo , Factores de Transcripción , Animales , Diferenciación Celular , División Celular , Linaje de la Célula , Señales (Psicología) , Proteínas de Unión al ADN/genética , Drosophila/citología , Drosophila/embriología , Drosophila/genética , Quinasas Janus , Ligandos , Masculino , Mutación , Proteínas Tirosina Quinasas/genética , Factores de Transcripción STAT , Transducción de Señal , Espermatocitos/citología , Espermatocitos/fisiología , Espermatogénesis , Células Madre/citología , Testículo/citología , Testículo/metabolismo , Transactivadores/genéticaRESUMEN
During development, many cells are specifically eliminated. Therefore, programmed cell death must be understood to fully elucidate embryogenesis. Retinoic acid (RA) and bone morphogenetic protein (BMP) 4 induce rapidly dividing P19 embryonal carcinoma cells to undergo apoptosis. RA alone minimally induces apoptosis, while BMP4 alone induces none. RA and BMP4 exposure also elevates the number of cells in the G1 phase of the cell cycle. Because many cell cycle proteins control both proliferation and apoptosis, we determined the role of these proteins in inducing apoptosis. Although the mRNA levels of cyclins D1 and D2 are reduced in cells undergoing apoptosis, the protein levels are not. In contrast, RA and BMP4 induce the Cdk inhibitor p27. This protein binds Cdk4 in RA- and BMP4-treated cells and inhibits Cdk4-dependent kinase activity. We used p27 antisense oligonucleotides to rescue the P19 cells from RA and BMP4 apoptosis thus proving that p27 is necessary. The Cdk4 substrate, retinoblastoma (Rb) protein, is also induced in apoptotic cells. Consistent with the decreased kinase activity of the apoptotic cells, this Rb protein is hypophosphorylated and presumably active. These data support the hypothesis that RA and BMP4 together induce the p27 protein leading to Rb activation and ultimately apoptosis.
Asunto(s)
Apoptosis , Proteínas Morfogenéticas Óseas/farmacología , Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogénicas , Tretinoina/farmacología , Proteínas Supresoras de Tumor , Proteína Morfogenética Ósea 4 , Carcinoma Embrionario , Ciclo Celular/efectos de los fármacos , Ciclina D , Quinasa 4 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/biosíntesis , Ectodermo/citología , Ectodermo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Unión Proteica , Proteína de Retinoblastoma/biosíntesis , Células Tumorales CultivadasRESUMEN
Mutagenesis of Vibrio cholerae with TnphoA, followed by screening for fusions that were activated under low-iron conditions, led to the identification of seven independent fusion strains, each of which was deficient in the ability to utilize ferrichrome as a sole iron source for growth in a plate bioassay and had an insertion in genes encoding products homologous to Escherichia coli FhuA or FhuD. Expression of the gene fusions was independent of IrgB but regulated by Fur. We report here a map of the operon and the predicted amino acid sequence of FhuA, based on the nucleotide sequence. Unlike those of the E. coli fhu operon, the V. cholerae ferrichrome utilization genes are located adjacent and opposite in orientation to a gene encoding an ATP-binding cassette transporter homolog, but this gene, if disrupted, does not affect the utilization of ferrichrome in vitro.
Asunto(s)
Proteínas de Escherichia coli , Ferricromo/metabolismo , Genes Bacterianos , Hierro/metabolismo , Operón , Vibrio cholerae/genética , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Receptores Virales/genética , Proteínas Recombinantes de Fusión , Homología de Secuencia de AminoácidoRESUMEN
The growth rate of malignant F9 embryonal carcinoma cells slows considerably following all-trans-retinoic acid-induced differentiation into benign parietal endoderm. To determine the mechanism of this process, we examined the expression of cyclins D1, D2, and D3 and the activity of their associated kinases. Cyclin D1 and D3 mRNA levels decreased during complete differentiation induced by all-trans-retinoic acid and dibutyryl cAMP, while the levels of cyclin D2 and the cyclin-dependent kinase (Cdk) inhibitor p27 mRNAs increased. Ultimately, terminally differentiated cells possessed 50% of the Cdk4-associated kinase activity observed in undifferentiated cells. Since numerous genes are differentially regulated during parietal endoderm differentiation, it is difficult to determine whether retinoic acid affects cell cycle gene expression directly or if these changes are caused by differentiation. We found that the retinoid X receptor (RXR)-selective agonists LG100153 and LG100268 significantly inhibited F9 cell growth without causing overt terminal differentiation as assessed by anchorage-independent growth and differentiation-associated gene expression. As seen in cells induced to differentiate by the RAR agonist all-trans-retinoic acid, RXR activation led to an increase in the number of cells in G1 phase. RXR agonists also sharply induced the levels of the Cdk regulatory subunits, cyclin D2 and D3. However, Cdk4-dependent kinase activity was reduced by RXR-selective retinoid treatment. These observations suggest that some retinoids can directly inhibit proliferation and regulate Cdk4-dependent kinase activity without inducing terminal differentiation.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Embrionario , Ciclina D1/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas , Receptores de Ácido Retinoico/genética , Factores de Transcripción/genética , Tretinoina/farmacología , Alitretinoína , Animales , Benzoatos/farmacología , Biomarcadores , Adhesión Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Ciclina D1/análisis , Ciclina D1/metabolismo , Ciclina D2 , Ciclina D3 , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/análisis , Ciclinas/genética , Ciclinas/metabolismo , Endodermo/citología , Ácidos Nicotínicos/farmacología , ARN Mensajero/análisis , Receptores de Ácido Retinoico/agonistas , Receptores X Retinoide , Células Madre/citología , Células Madre/enzimología , Tetrahidronaftalenos/farmacología , Factores de Transcripción/agonistas , Transcripción Genética/fisiología , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/enzimologíaRESUMEN
We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.
Asunto(s)
Toxina del Cólera/genética , Exotoxinas/genética , Familia de Multigenes , Vibrio cholerae/genética , Secuencia de Aminoácidos , Supervivencia Celular/efectos de los fármacos , Mapeo Cromosómico , Cromosomas Bacterianos , Secuencia de Consenso , Exotoxinas/química , Exotoxinas/toxicidad , Genoma Bacteriano , Humanos , Datos de Secuencia Molecular , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Eliminación de Secuencia , Células Tumorales Cultivadas , Vibrio cholerae/clasificación , Vibrio cholerae/virologíaRESUMEN
Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transcripción Genética , Factor de Crecimiento Transformador beta/genética , Tretinoina/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Proteína Morfogenética Ósea 2 , Carcinoma Embrionario/genética , Carcinoma Embrionario/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Drosophila , Genes Reporteros , Datos de Secuencia Molecular , Plásmidos , ARN Mensajero/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , beta-Galactosidasa/genéticaRESUMEN
This is a summary of the present status of The MYTHSEEKER Project. The first section provides a brief synopsis of the project's purpose and approach. The second section summarizes present activity in a number of directions-including decisions that are shaping development. The third section outlines the next steps for development programming, the initial software product, and a communications network of MYTHSEEKER Centers. Some extended viewpoints are mentioned.
RESUMEN
Some growth factors, for example, members of the transforming growth factor-beta family, can induce apoptosis in a variety of cells. Retinoic acid (RA) also causes apoptosis in several malignant cell types. We have previously demonstrated that, although BMP2 or BMP4 cannot induce apoptosis alone, BMP2 or BMP4 and RA synergize to induce apoptosis in 95% of P19 embryonal carcinoma cells within 4 days of treatment. Such treatment also prevents neuronal differentiation of these cells. Retinoids exert their many effects through any of six distinct nuclear receptors. These retinoid-activated transcription factors directly regulate genes involved in cellular response such as apoptosis. Complete understanding of how BMP and RA specifically induce cell death requires identification of the retinoid receptors controlling apoptosis. By using receptor-selective retinoid agonists and antagonists, we have obtained evidence suggesting that activation of RAR alpha or gamma is sufficient to induce apoptosis in BMP4-treated cells.
Asunto(s)
Apoptosis/fisiología , Proteínas Morfogenéticas Óseas/farmacología , Células Madre Neoplásicas/metabolismo , Receptores de Ácido Retinoico/metabolismo , Retinoides/farmacología , Animales , Proteína Morfogenética Ósea 4 , Línea Celular , Células Madre de Carcinoma Embrionario , Regulación Enzimológica de la Expresión Génica , Ratones , Células Madre Neoplásicas/citología , ARN Mensajero/análisis , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptor alfa de Ácido Retinoico , Receptores X Retinoide , Factores de Transcripción/agonistas , Transglutaminasas/genética , Receptor de Ácido Retinoico gammaRESUMEN
We have previously cloned a novel guanine nucleotide-binding protein (G-protein)-coupled receptor, H218, that has sequence similarity to a lysophosphatidic acid receptor, edg2. We present here Northern analysis indicating that the H218 mRNA is expressed in undifferentiated F9 embryonal carcinoma cells. The H218 message is down-regulated and its stability is decreased during retinoic acid- and dibutyryl cAMP-induced differentiation. Treatment by various receptor-selective retinoids indicated that retinoic acid receptor beta or gamma signaling, but not retinoid X receptor activation, is required for the down-regulation of H218 mRNA. Activation of the H218 receptor may contribute to the phenotype of undifferentiated F9 embryonal carcinoma cells.
Asunto(s)
Carcinoma Embrionario/patología , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Receptores de Superficie Celular/biosíntesis , Receptores Acoplados a Proteínas G , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Animales , Benzoatos/farmacología , Bucladesina/farmacología , Diferenciación Celular/efectos de los fármacos , Cromanos/farmacología , Proteínas de Unión al GTP/fisiología , Ratones , Naftalenos/farmacología , Proteínas de Neoplasias/genética , Fenotipo , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Receptores de Superficie Celular/genética , Receptores Lisofosfolípidos , Receptores de Ácido Retinoico/clasificación , Receptores de Ácido Retinoico/efectos de los fármacos , Retinoides/farmacología , Tetrahidronaftalenos/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
This chapter outlines the use in psychotherapy and medical diagnosis of an intelligent software system that helps clients to explore Personal Myth within virtual reality environments. Patented MYTHSEEKER software will allow clients to work with mythic analogues of lifeshapes and aspirations. This can help to focus therapy directions, find ways to participate with the person's world, and allow a kind of personal expression not previously possible. The software phases of assessment, facilitation, and enaction are described by which the client is assisted to explore systems of mythology or spirituality (called Depth Systems) that are traditional, ancient or newly-arising. The client builds a Personal Depth System representing Personal Myth, based on experiencing other Depth Systems, which can itself be experienced in the virtual environment. This paper outlines our methodology and technology to realize these operations. Space limitations prevent further description in the present chapter of MYTHSEEKER software technology or psychotherapy scenarios of involvement.
Asunto(s)
Simulación por Computador , Procesamiento de Imagen Asistido por Computador/instrumentación , Psicoterapia/instrumentación , Programas Informáticos , Terapia Asistida por Computador/instrumentación , Interfaz Usuario-Computador , Inteligencia Artificial , Aspiraciones Psicológicas , Humanos , Individualidad , Motivación , Medio SocialRESUMEN
We evaluated a spontaneous mutant of Vibrio cholerae, which was avirulent in an infant mouse and had reduced expression of cholera toxin and TcpA in response to environmental signals. The toxR, toxS and toxT genes in the mutant were normal, but transcription of toxT was absent. A plasmid expressing wild-type tcpP and tcpH complemented the mutant. The mutation resulted from a frameshift in a string of nine G residues within tcpH; similar slipped-strand mutations in tcpH arose at a frequency of 10(-4) during overnight growth and in the majority of colonies by the end of 5 days of growth in ToxR-inducing conditions. Transcription of tcpPH was regulated by temperature and pH independently of ToxR or ToxT. These results suggest that TcpH couples environmental signals (temperature and pH) to expression of the ToxR regulon, and provide a model for phase variation in the co-ordinate expression of cholera virulence factors.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Proteínas Fimbrias , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana , Regulón , Factores de Transcripción/genética , Vibrio cholerae/genética , Aglutinación , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Cólera/microbiología , Toxina del Cólera/biosíntesis , Toxina del Cólera/genética , ADN Bacteriano , Mutación del Sistema de Lectura , Prueba de Complementación Genética , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Fenotipo , Transcripción Genética , Vibrio cholerae/patogenicidad , VirulenciaAsunto(s)
Apoptosis/fisiología , Ciclo Celular/fisiología , Retinoides/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , División Celular/fisiología , Humanos , Retinoides/farmacología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Tretinoina/fisiologíaRESUMEN
This review focuses on known genes whose expression may be perturbed by teratogens during early embryogenesis (preorganogenesis). Teratogens may disrupt embryogenesis by modifying positional information. Genes controlling positional information include those specifying the primary body axes: anterior-posterior, dorsal-ventral, or left-right. These genes often encode transcription factors, whose regulation or activation can stimulate aberrant tissue differentiation and morphogenesis. Alternatively, teratogens may directly affect cell differentiation, proliferation, or apoptosis. Hydrophilic signalling molecules such as growth factors and hydrophobic molecules such as retinoids regulate these processes. The signalling pathways activated often induce the coordinate regulation of tissue specific gene expression. In addition to modifying individual signalling pathways, teratogens can synergize with or antagonize the effects of other teratogens through inappropriate interactions between signal transduction pathways. Since teratogens may often directly or indirectly perturb the expression of known or as yet undescribed developmentally critical genes, this review also provides a short description of techniques to identify genes whose expression is altered by teratogens.
Asunto(s)
Embrión de Mamíferos/efectos de los fármacos , Desarrollo Embrionario y Fetal/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Teratógenos/toxicidad , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/efectos de los fármacos , Desarrollo Embrionario y Fetal/genética , Genes Homeobox/efectos de los fármacos , Técnicas Genéticas , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/embriología , Transducción de Señal/efectos de los fármacos , Teratógenos/farmacología , Factores de Transcripción/fisiología , Vertebrados/embriología , Vertebrados/genéticaRESUMEN
Retinoic acid (RA) affects the response of many cells to growth factors, including the bone morphogenetic proteins (BMPs). The BMPs are members of the TGF-beta, family of growth factors, originally identified by their bone-inducing activities. Their widespread expression suggests many roles other than that in osteogenesis. Because RA modulates the cell's response to growth factors, this may be a means by which the retinoids exert some of their known teratogenic effects. One such cellular response may be apoptosis. While apoptosis is required for normal development, the location and timing of its induction must be carefully controlled. Recently, several TGF-beta family members have been implicated in the induction of apoptosis in certain cell types. We show here, using P19 embryonal carcinoma cells, that the combination of RA and BMP2 or BMP4 synergistically induces apoptosis in 40% of the population within 24 hr. In contrast, RA alone induces apoptosis in only 10-15% of the population and each of the BMPs alone minimally induces apoptosis. Apoptosis depends on the dose of both the RA and the BMP as well as on new protein synthesis. Further, the induction of apoptosis prevents the formation of fully differentiated neurons and glial cells and instead leads to primarily smooth muscle cell differentiation. These results suggest that some of the malformations caused by retinoids may be due to the induction of inappropriate apoptosis in cells exposed to BMPs.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Morfogenéticas Óseas/farmacología , Carcinoma Embrionario/patología , Queratolíticos/farmacología , Factor de Crecimiento Transformador beta , Tretinoina/farmacología , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Sinergismo Farmacológico , Citometría de Flujo , Células Tumorales CultivadasRESUMEN
Iron is an essential nutrient to support the growth of most bacterial species. However, iron is not easily available to microorganisms infecting mammalian hosts, because it is largely sequestered by iron-binding proteins, such as transferrin or lactoferrin, or complexed to heme. In response to environmental iron stress, Vibrio cholerae produces the siderophore vibriobactin as well as a number of iron-induced outer membrane proteins. Previous data on the role of iron acquisition systems for the intraintestinal growth of mucosal pathogens such as V. cholerae are conflicting. In this report, we isolated mutants of V. cholerae with TnphoA fusions in each of viuA, hutA, and irgA, as well as strains mutant in each pair of these genes and all three simultaneously, to analyze the role of these iron-induced outer membrane protein receptors for in vivo growth of V. cholerae. The fusion between hutA and TnphoA in a single copy on the chromosome allowed the study of in vitro regulation of hutA in response to iron, fur, and irgB; transcription of hutA was tightly iron regulated (70-fold) and dependent on a functional Fur but did not require IrgB. To investigate the effects of mutations in these iron-induced outer membrane proteins on in vivo growth, we inoculated ileal loops in a rabbit model of infection. This avoids exposure of organisms to the potential killing effects of gastric acid, allows several logarithmic increases in growth in the in vivo environment, and facilitates direct comparison of multiple strains in the same animal to avoid any differences between animals. We grew each mutant to be tested in competition with the wild-type strain in the same loop, to provide an internal control. We confirmed that the inocula for these experiments were grown under conditions of iron stress prior to in vivo inoculation, by measuring the alkaline phosphatase activity of the iron-regulated fusion in each strain. The results confirmed that mutation of irgA produced a much more substantial in vivo growth defect than mutation of either hutA or viuA alone. Double mutants of irgA with either viuA or hutA, or the strain mutant in all three genes, showed an in vivo growth defect comparable to the strain mutant in irgA only, suggesting that mutation of irgA was the most relevant for in vivo growth. The strain mutant in both hutA and viuA was also markedly impaired for in vivo growth, suggesting that mutation of both of these iron uptake systems simultaneously can also produce a substantial in vivo growth defect.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Hierro/metabolismo , Proteínas de Transporte de Membrana , Vibrio cholerae/crecimiento & desarrollo , Vibrio cholerae/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico Activo , Proteínas Portadoras/genética , Expresión Génica , Genes Bacterianos , Hemo/metabolismo , Masculino , Ratones , Mutagénesis Insercional , Mutación , Conejos , Vibrio cholerae/genéticaRESUMEN
The effect of retinoids on malignant cells and embryos indicates that retinoids influence the expression of growth factors or alter the response of cells to growth factors. The bone morphogenetic proteins, Bmp-2 and Bmp-4, are candidates for such growth factors because retinoic acid (RA) treatment of F9 embryonal carcinoma cells induced Bmp-2 mRNA, while simultaneously repressing Bmp-4 levels. Also, recombinant Bmp-2 affected the growth and differentiation of these cells. Regulation of each gene was concentration dependent and required continuous RA treatment. The short half-lives of the Bmp-2 (75 +/- 11 min) and Bmp-4 (70 +/- 4 min) mRNAs suggest that their abundance is primarily controlled at the transcriptional level. To determine which RA receptor (RAR) controls bmp-2 and bmp-4 expression, F9 cells were exposed to various receptor-selective retinoids. RAR alpha- and gamma-selective retinoids induced Bmp-2 and repressed Bmp-4 equally as well as all-trans RA. In contrast, a RAR beta-selective retinoid had little effect on Bmp-2 induction but repressed Bmp-4. A RAR alpha-selective antagonist inhibited all-trans RA stimulation of Bmp-2, although not as dramatically as a RAR beta gamma-selective antagonist. No differences were observed between Bmp levels in all-trans RA and 9-cis RA-treated cells, indicating that the RXRs play little part in controlling these genes. The results are consistent with RAR alpha and gamma-controlled Bmp-2 and Bmp-4 regulation.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Sustancias de Crecimiento/genética , Receptores de Ácido Retinoico/metabolismo , Tretinoina/farmacología , Animales , Benzoatos/farmacología , Northern Blotting , Bucladesina/farmacología , Células Madre de Carcinoma Embrionario , Regulación Neoplásica de la Expresión Génica/fisiología , Ratones , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/ultraestructura , ARN Mensajero/metabolismo , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptor alfa de Ácido Retinoico , Retinoides/farmacología , Tetrahidronaftalenos/farmacología , Teofilina/farmacología , Receptor de Ácido Retinoico gammaRESUMEN
Vitamin A is required for maintaining healthy epithelial tissues, e.g., skin and gut, normal reproductive capacity, and vision. Vitamin A deficiency also causes premalignant changes in epithelial tissues. These observations led to the use of retinoids (vitamin A-related compounds) in treating various skin diseases and more recently to inhibit cancer growth. Retinoids are extremely teratogenic to developing vertebrate embryos, a fact which limits their clinical usefulness. In particular, specific malformations of skeletal structures are often observed; for instance, retinoic acid induces craniofacial and limb deformities in avian and mammalian embryos. Recent technological advances have increased our understanding of how retinoids affect vertebrate development. Some newly discovered mechanisms underlying these actions are reviewed.
Asunto(s)
Desarrollo Embrionario y Fetal/fisiología , Retinoides/farmacología , Vertebrados/fisiología , Vitamina A/fisiología , Animales , Desarrollo Embrionario y Fetal/genética , Humanos , Morfogénesis/genética , Morfogénesis/fisiología , Vertebrados/genéticaRESUMEN
Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including beta-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low- and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B. fragilis 638 cepA mutant. The results of beta-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.
Asunto(s)
Bacteroides fragilis/enzimología , Cefalosporinasa/genética , Elementos Transponibles de ADN , Secuencia de Aminoácidos , Bacteroides fragilis/genética , Secuencia de Bases , Cefalosporinasa/química , Cefalosporinasa/metabolismo , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de SecuenciaRESUMEN
Bacteroides fragilis CS30 is a clinical isolate resistant to high concentrations of benzylpenicillin and cephaloridine but not to cephamycin or penem antibiotics. beta-Lactam resistance is mediated by a chromosomally encoded cephalosporinase produced at a high level. The gene encoding this beta-lactamase was cloned from genomic libraries constructed in Escherichia coli and then mated with B. fragilis 638 for identification of ampicillin-resistant (Apr) strains. Apr transconjugants contained a nitrocefin-reactive protein with the physical and enzymatic properties of the original CS30 isolate. The beta-lactamase gene (cepA) was localized by deletion analysis and subcloned, and its nucleotide sequence was determined. The 903-bp cepA open reading frame encoded a 300-amino-acid precursor protein (predicted molecular mass, 34,070 Da). A beta-lactamase-deficient mutant strain of B. fragilis 638 was constructed by insertional inactivation with the cepA gene of CS30, demonstrating strict functional homology between these chromosomal beta-lactamase genes. An extensive comparison of the CepA protein sequence by alignment with other beta-lactamases revealed the strict conservation of at least four elements common to Ambler class A. A further comparison of the CepA protein sequence with protein sequences of beta-lactamases from two other Bacteroides species indicated that they constitute their own distinct subgroup of class A beta-lactamases.